Method and apparatus for determining substrate concentration and reagent for determining substrate concentration

ABSTRACT

The present invention relates to a method, a reagent and an apparatus for determining a substrate concentration based on an amount of hydrogen peroxide generated from a substrate. In the present invention, a suppressing agent for suppressing a reaction between the hydrogen peroxide and an inhibitor is added. As the suppressing agent, an azide compound such as sodium azide or a nitrite compound such as sodium nitrite is used. In the invention, a supporting electrolyte, such as sodium chloride or potassium chloride may be further added.

TECHNICAL FIELD

The present invention relates to a method, an apparatus and a reagent for carrying out the measurement of a substrate such as glucose.

BACKGROUND ART

There is a method called an enzyme-electrode method, which is a method for determining a glucose concentration. According to the method, information correlating with a glucose concentration in a specimen is outputted to an electrode in contact with the specimen, and the glucose concentration is calculated based on the output. In an example of the method, electrons are removed at an electrode from hydrogen peroxide generated by oxidizing glucose with an enzyme, and a glucose concentration is calculated based on a current value measured at the time in the electrode (for example, see the Patent Document 1)

When the glucose concentration is determined, blood may be used as the specimen in some cases. In a case where hemolysis in hemocyte occurs, hemoglobin may be included in the specimen whether the whole blood is used or blood serum or plasma is used.

However, the hemoglobin easily reacts with the hydrogen peroxide. Therefore, the hemoglobin included in the specimen inhibits the reaction between the hydrogen peroxide and the electrode. As a result, the electrode output tends to show a lower value as the reaction between the hemoglobin and the hydrogen peroxide advances in the presence of the hemoglobin, which makes it difficult to obtain high determination accuracy.

It is not avoidable for the specimen to include the hemoglobin by preventing the occurrence of hemolysis of the hemocyte. This is, however, not a realistic approach because it is necessary to pay a meticulous attention during the transportation, preservation and pretreatment of the blood, which demands burdensome handling steps, and the likelihood of occurrence of hemolyis varies depending on an analyte.

[Patent Document 1] JP-B-H07-37991 DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

An object of the present invention is to improve determination accuracy by controlling any influences from an inhibitor when the concentration of a substrate, for example, glucose in a specimen such as blood, is determined without any complicated and time-consuming handling steps.

Means for Solving the Problems

According to a first aspect of the present invention, there is provided a method for determining a substrate concentration based on the amount of hydrogen peroxide generated from a substrate, wherein a suppressing agent for suppressing a reaction between the hydrogen peroxide and an inhibitor is allowed to coexist.

According to a second aspect of the present invention, there is provided a concentration determination apparatus comprising a hydrogen peroxide electrode for determining the concentration of hydrogen peroxide generated from a substrate, wherein a suppressing agent for suppressing a reaction between the hydrogen peroxide and an inhibitor is supplied.

According to a third aspect of the present invention, there is provided a reagent for determining a substrate concentration based on the amount of hydrogen peroxide generated from a substrate, wherein the reagent includes a suppressing agent for suppressing a reaction between the hydrogen peroxide and an inhibitor.

As the suppressing agent, for example, a compound which transforms the hemoglobin as an inhibitor into methemoglobin is used, and at least one selected from azide compounds; nitrites; ferricyanides; peroxides; and permanganates is used as the compound which transforms the hemoglobin into the methemoglobin.

Examples of the azide compound to be used include sodium azide; 4-dodecylbenzenesulfonyl azide; 4-acetylaminobenzenesulfonyl azide; diphenylphosphoric azide; iron azide; hydrogen azide; lead azide; mercury azide; copper azide; and silver azide. The concentration of the azide compound in the determination system is preferably 0.001 to 1.000 wt %, and more preferably 0.010 to 0.100 wt %.

Examples of the nitrite to be used include sodium nitrite and potassium nitrite. Examples of the ferricyanide to be used include potassium ferricyanide, examples of the peroxide to be used include barium peroxide, and examples of the permanganate to be used include potassium permanganate. When any of the listed salts is used as the suppressing agent, the concentration of the suppressing agent in the determination system is, for example, preferably 0.001 to 1.000 wt %, and more preferably 0.010 to 0.100 wt %.

In the present invention, preferably, a supporting electrolyte is further allowed to coexist (to be added). Examples of the supporting electrolyte to be used include sodium chloride and potassium chloride, and sodium chloride is preferably used as the supporting electrolyte. The concentration of the supporting electrolyte is preferably 0.010 to 2.000 wt %, and more preferably 0.050 to 0.500 wt %.

Examples of the substrate include glucose and lactate. The substrate is included in a biochemical specimen such as blood, urine or saliva.

According to the method and apparatus of the present invention, an enzyme-electrode method is employed to determine the amount of the hydrogen peroxide.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described below referring to the drawings.

A concentration determination apparatus 1 illustrated in FIG. 1 is configured to measure a specimen adjusted in a specimen preparation mechanism 2 using a measurement mechanism 3.

The specimen preparation mechanism 2 is a mechanism for preparing a specimen from an analyte, and comprises a nozzle 20, a reagent bottle 21, and a diluting/mixing tank 22.

The nozzle 20 is a nozzle for supplying an analyte into the diluting/mixing tank 22. Examples of the analyte to be used are biochemical specimens such as blood, urine and saliva, or diluents obtained therefrom. In a case where blood is used as the specimen, any of the whole blood and blood serum or plasma can be used.

The reagent bottle 21 retains therein a reagent for diluting an analyte or washing the diluting/mixing tank 22. The reagent bottle 21 is connected to the diluting/mixing tank 22 with a pipe 23 interposed therebetween. A pump 24 is provided at an intermediate position in the pipe 23, and a configuration is given such that a force generated by the pump 24 serves to supply the reagent retained in the reagent bottle 21 into the diluting/mixing tank 22.

A material including a diluent and a suppressing agent is used as the reagent.

Examples of the diluent include a buffer solution. The buffer solution is not particularly limited as far as it can adjust the reaction pH of a substrate in a targeted range, and a phosphate, for example, can be used. The concentration of the buffer solution in the reagent is set to, for example, 0.0001 to 0.1000 M.

The suppressing agent serves to suppress a reaction between hydrogen peroxide as the substrate and an inhibitor. In a case where the inhibitor is hemoglobin, a compound which transforms the hemoglobin into methemoglobin, for example, at least one selected from: azide compounds; nitrites; ferricyanide; peroxides; and permanganates is used as the suppressing agent.

Examples of the azide compound to be used include sodium azide (NaN₃); sodium azide; 4-dodecylbenzenesulfonyl azide; 4-acetylaminobenzenesulfonyl azide, diphenylphosphoric azide; iron azide; hydrogen azide; lead azide; mercury azide; copper azide; and silver azide. The concentration of the azide compound included in the reagent is, for example, 0.001 to 1.000 wt %, and preferably 0.010 to 0.100 wt %.

Examples of the nitrite to be used are sodium nitrite and potassium nitrite. Examples of the ferricyanide to be used include potassium ferricyanide, examples of the peroxide to be used include barium peroxide, and examples of the permanganate to be used include potassium permanganate. When any of the listed salts is used as the suppressing agent, the concentration of the suppressing agent in the determination system is, for example, preferably 0.001 to 1.000 wt %, and more preferably 0.010 to 0.100 wt %.

The reagent may further include a supporting electrolyte. Examples of the supporting electrolyte to be used are sodium chloride and potassium chloride. The concentration of the supporting electrolyte in the reagent is, for example, 0.010 to 2.000 wt %, and preferably 0.050 to 0.500 wt %.

The diluting/mixing tank 22 is a tank for providing a place where an analyte is diluted so that a specimen is prepared. The diluting/reaction tank 22 is configured such that the analyte is supplied by means of the nozzle 20, and the reagent is supplied from the reagent bottle 21. The diluting/mixing tank 22 comprises therein an agitator 25. When the agitator 25 is rotated by a stirrer 26, the analyte and the reagent are mixed with each other. The diluting/mixing tank 22 is further connected to an enzyme electrode 30 of the measurement mechanism 3 with a pipe 27 interposed therebetween. A pump 28 is provided at an intermediate position in the pipe 27, and a configuration is given such that a force generated by the pump 28 serves to supply a specimen prepared in the diluting/mixing tank 22 to the enzyme electrode 30 of the measurement mechanism 3.

The measurement mechanism 3 comprises the enzyme electrode 30, a power supply 31 and a current value measuring unit 32.

The enzyme electrode 30 outputs an electrophysical quantity corresponding to the amount of electrons transferred to and received from a substrate in a specimen. As illustrated in FIG. 2, the enzyme electrode 30 comprises a substrate selective transmission film 33, an enzyme immobilized film 34, a hydrogen peroxide selective transmission film 35, and a hydrogen peroxide electrode 36.

The substrate selective transmission film 33 is a film for selectively supplying a substrate in a specimen to the enzyme immobilized film 34. The substrate selective transmission film 33 is selected depending on the type of a substrate, and any of various conventionally available films may be used as the substrate selective transmission film 33.

The enzyme immobilized film 34 is a film for generating hydrogen peroxide by oxidizing a substrate, and is configured to include an oxidase. The oxidase used in the present invention is selected depending on the type of a substrate. Examples of the substrate to be measured by the concentration determination apparatus 1 include glucose and lactate, and glucose oxidase or lactate oxidase can be mentioned as the oxidase.

The hydrogen peroxide selective transmission film 35 is a film for selectively supplying hydrogen peroxide to the hydrogen peroxide electrode 36. Examples of the hydrogen peroxide selective transmission film 35 to be used include acetylcellulose-based films and polyarylamine-based films.

The hydrogen peroxide electrode 36 outputs an electrical signal corresponding to the amount of the supplied hydrogen peroxide, in other words, the concentration of the substrate. The hydrogen peroxide electrode 36 to be used may be, for example, an electrode in which platinum is used as an anode 37, while silver is used as a cathode 38.

The power supply 31 illustrated in FIG. 1 applies a voltage to the enzyme electrode 30 (hydrogen peroxide electrode 36). A direct-current power supply, for example, is used as the power supply 31, and the voltage applied to the enzyme electrode 30 (hydrogen peroxide electrode 36) is set to, for example, 0.1 to 1.0 V.

The current value measuring unit 32 is a unit for measuring the amount of electrons transferred to and from the hydrogen peroxide electrode 36 and the hydrogen peroxide as a current value. The current value is intermittently measured by the current value measuring unit 32, and a measurement interval is set to, for example, 50 to 200 msec.

Next is described an operation of the concentration determination apparatus 1 in a case where glucose in the whole blood is a target of the determination.

When the concentration of the glucose in the whole blood is determined, the whole blood and the reagent are supplied into the diluting/mixing tank 22. The whole blood is supplied into the diluting/mixing tank 22 by means of the nozzle 20, and the amount of the whole blood to be supplied is set to, for example, 4 to 20 μL. On the other hand, the reagent is supplied into the diluting/mixing tank 22 by means of the pump 24, and the amount of the reagent to be supplied is set to, for example, approximately 100 times as much as the amount of the whole blood.

When the whole blood and the reagent are supplied in the concentration determination apparatus 1, they are agitated by the agitator 25 in the diluting/mixing tank 22. Because the amount of the reagent to be supplied is set to approximately 100 times as much as the amount of the whole blood to be supplied, the pH of the buffer solution in the diluting/mixing tank 22 and the concentrations of the suppressing agent and the supporting electrolyte are substantially retained to be equal to the pH and the concentrations in the reagent. After that, the prepared specimen is supplied to the enzyme electrode 30 by means of the pump 28.

In the enzyme electrode 30, the glucose transmits through the substrate selective transmission film 33 and is then supplied to the enzyme immobilized film 34. In the enzyme immobilized film 34, the glucose of the oxidase (glucose dehydrogenase) is oxidized, and gluconic acid and hydrogen peroxide are generated as illustrated in the following reaction formula (1). The hydrogen peroxide transmits through the hydrogen peroxide selective transmission film 35 and is then supplied to the hydrogen peroxide electrode 36. In the hydrogen peroxide electrode 36, the reaction represented by the following reaction formula (2) occurs by the application of the voltage by the power supply 31, and the hydrogen peroxide supplies electrons to the anode 37 to be decomposed into oxygen and hydrogen ions. At the time, the electrons supplied to the anode 37 generate a current flow between the anode 37 and the cathode 38, and the current generated at the time is measured by the current value measuring unit 32. On the other hand, in the cathode 38, the electrons from the anode 37 generate the reaction represented by the following reaction formula (3), whereby water is produced.

In the anode 38, the reductive reaction of the hydrogen peroxide (H₂O₂) occurs. In the presence of oxyhemoglobin (Fe (II)) in the diluting/mixing tank 22, the anodic reaction and the oxidizing reaction of deoxyhemoglobin (oxy-Hb (Fe II)) into methemoglobin (met-Hb (Fe (III)) compete with each other as illustrated in the following reaction formula (4).

In a case where the competing reactions occur, the hydrogen peroxide, which is supposed to be consumed in the anode 37, is consumed by the deoxyhemoglobin. Accordingly, the current value measured by the current value measuring unit 32 falls below an expected value to be fundamentally obtained. In the specimen including hemocyte, in particular, the pH in the vicinity of a hemocyte membrane tends to be lowered because gluconic acid is generated, and the oxyhemoglobin releases oxygen and is thereby transformed into the deoxyhemoglobin with accompanying the decrease of the pH. Therefore, it is considered that the production of the deoxyhemoglobin is increased in accordance with the progress of the oxidizing reaction of the glucose. Thus, the deoxyhemoglobin which inhibits the oxidizing reaction of the hydrogen peroxide in the anode 37 is generated in accordance with the progress the oxidizing reaction of the glucose (gluconic acid is generated), which is considered a probable reason why the measured oxidation current is smaller than the true value.

On the other hand, when an oxide such as an azide compound or nitrite is allowed to coexist as the suppressing agent, the oxidizing reaction of the deoxyhemoglobin (Fe (II)) into the methemoglobin (Fe (III)) occurs by the oxidative effect of the suppressing agent as illustrated in the following reaction formula (5).

Therefore, the reaction of the hydrogen peroxide with the deoxyhemoglobin is suppressed when the suppressing agent is allowed to coexist in the specimen. As a result, the current value measured by the current value measuring unit 32 can be prevented from decreasing, and the variability of the measured response current value can be suppressed in the concentration determination apparatus 1. Thus, the reduction of the result of the concentration determination can be suppressed, which leads to the improvement of reproducibility, and the accuracy and reliability in the measurement can be thereby improved.

The effect thus far described can also be obtained in any other system where hydrogen peroxide is generated by a substrate and the hydrogen peroxide and an anode are reacted with each other so that a substrate concentration is determined, without limiting to a case where an inhibitor is deoxyhemoglobin or a substrate is glucose.

Further, ionic strength in the reaction tank 20 can be increased when the supporting electrolyte such as sodium chloride is included in the reagent, and the oxidation current obtained in the current value measuring unit 32 can be thereby stabilized. Thus, the inclusion of the supporting electrolyte can further improve the measurement reproducibility.

The present invention is not limited to the embodiment described so far, and can be variously modified. For example, the enzyme electrode may be configured to be fixated in the diluting/mixing tank, and the substrate concentration may be measured according to the batch system.

Further, the diluting/mixing tank is omitted as in the concentration determination apparatus 1 illustrated in FIG. 3, and a configuration may be given such that the reagent of the reagent bottle 21 is continuously supplied to the enzyme electrode 30, and the analyte and specimen are injected from an injection valve 4.

Example 1

In this example, the influence of sodium azide onto a response value when a glucose concentration in a specimen is determined by means of a hydrogen peroxide electrode was studied.

(Specimen)

The whole blood in which the glucose concentration is adjusted to 100 mg/dL was used as the specimen.

(Reagent)

As a reagent, a sample 1 in which NaCl is added by 0.234 wt % to “61H” (manufactured by ARKRAY, Inc.), and a sample 2 in which NaN₃ is further added by 0.02 wt % to the sample 1 were respectively diluted 100 times and then supplied into a reaction tank.

(Measurement of Response Value)

The response value was measured by ADAMS GLUCOSE GA-117 (manufactured by ARKRAY, Inc.) as an oxidation current obtained in a GOD-immobilized hydrogen peroxide electrode when the specimen and reagent were supplied into the reaction tank. The voltage to be applied to the hydrogen peroxide electrode was set to 650 mV, and a response current was measured at 50-msec intervals. An operation in which the specimen and reagent were supplied to the reaction tank and retained therein for a certain period of time and then replaced with another specimen and reagent, which were retained for a certain period of time is set as one round, and the response current was continuously measured in three rounds in total. At intervals between the rounds, the reaction tank and the pipe were washed for a certain period of time. The measurement results of response current value, which were converted into voltage values, were illustrated in the drawings, the specimen 1 in FIG. 4, while the specimen 2 in FIG. 5.

As is known when FIGS. 4 and 5 are compared to each other, the specimen 1 not adding NaN₃ showed a result that maximum response values were relatively low and also variable without any distinct peak. It is considered that the low values were obtained because the amount of hydrogen peroxide measured in a hydrogen peroxide electrode was lessened when the hydrogen peroxide generated upon oxidizing glucose reacts with hemoglobin.

On the other hand, the result of the specimen 2 in which NaN₃ was added showed maximum values larger than those of the specimen 1, and a stabilized time course of the response value. More specifically, in the specimen 2 in which NaN₃ was added, it is considered that hemoglobin and NaN₃ react with each other, which is expected to prevent the amount of hydrogen peroxide measured in a hydrogen peroxide from decreasing.

It is found from these results that the measurement accuracy and reproducibility are improved when NaN₃ is added to the reaction system since the increase of the response value is observed and the maximum values thereof are stabilized.

Example 2

In this example, the influences of sodium azide and sodium nitrite onto a response value when a glucose concentration in a specimen is determined by means of a hydrogen peroxide electrode were studied.

(Specimen)

The whole blood in which the glucose concentration is adjusted to 100 mg/dL was used as the specimen.

(Reagent)

As a reagent, a sample 3 in which NaCl is added by 0.234 wt % to “61H” (manufactured by ARKRAY, Inc.), a sample 4 in which NaN₃ is further added by 0.02 wt % to the sample 3, and a sample 5 in which sodium nitrite is added by 0.02 wt % to the sample 3 were respectively diluted 100 times and then supplied into a reaction tank.

(Measurement of Response Value)

The response value was measured by ADAMS GLUCOSE GA-117 (manufactured by ARKRAY, Inc.) as an oxidation current obtained in a GOD-immobilized hydrogen peroxide electrode when the specimen and reagent were supplied into the reaction tank. The voltage to be applied to the hydrogen peroxide electrode was set to 650 mV, and a response current was measured at 50-msec intervals. The response current was continuously measured in a sequence of operations in which the specimen and reagent were supplied into the reaction tank and agitated for a certain period of time and then the agitation was terminated. The measurement results of the response current values, which were converted into voltage values, were illustrated in the drawings, the specimen 3 in FIG. 6A, the specimen 4 in FIG. 6B, and the specimen 5 in FIG. 6C.

As is known from FIG. 6A, the specimen 3 in which NaN₃ and sodium nitrite were not added showed a deteriorated sensitivity in a while after the agitation was terminated. It is considered that it is because the oxidizing reaction of deoxyhemoglobin (oxy-Hb (Fe II)) into methemoglobin (met-Hb (Fe (III)) advances, and the anodic reaction of hydrogen peroxide is inhibited.

On the other hand, the sensitivity of the specimen 4 in which NaN₃ was added increased even after the agitation was terminated within the measurement range set in the present example as is known from FIG. 6B, while the sensitivity of the specimen 5 in which sodium nitrite was added inclined to approach asymptotically to a certain value after the agitation was terminated within the measurement range set in the present example as is known from FIG. 6A. More specifically, the deterioration of the sensitivity was not observed in a case where NaN₃ and sodium nitrite were added, which is considered that NaN₃ and sodium nitrite suppress the oxidizing reaction of deoxyhemoglobin (oxy-Hb (Fe II)) into methemoglobin (met-Hb (Fe (III)).

Example 3

In this example, the influence of NaCl in a reagent onto a response value was studied.

As the reagent, “61H” (manufactured by ARKRAY, Inc.) was prepared as a sample 6, and a sample 7 in which NaCl was added to the sample 6 by 0.234 wt % were used.

A physiological saline including NaCl of 0.9 wt % was diluted 100 times with the sample 6 or 7, and the obtained solution was used as the specimen.

The response value was measured in a manner similar to Example 1. The measurement results of response current values, which were converted into voltage values, were illustrated in the drawings, the sample 6 in FIG. 7, while the sample 4 in FIG. 8.

As is known from FIGS. 7 and 8, the response value was instable in the sample 6 in which NaCl was not added, while the response value was stable in the sample 7 in which NaCl was added. That is, it is considered that NaCl is likely to stabilize the basis of the response current value.

Therefore, it is considered that the response value is stabilized and the determination accuracy and reproducibility are improved when NaCl is added to the reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sectional view schematically illustrating a structure of a concentration determination apparatus according to the present invention.

FIG. 2 is a sectional view schematically illustrating a structure of an enzyme electrode provided in the concentration determination apparatus illustrated in FIG. 1.

FIG. 3 is a piping arrangement drawing for describing another example of the concentration determination apparatus according to the present invention.

FIG. 4 is a graph showing the measurement result of a response current in the whole blood in a case where sodium azide is not added in Example 1.

FIG. 5 is a graph showing the measurement result of a response current in the whole blood in a case where sodium azide is added in Example 1.

FIG. 6A is a graph showing the measurement result of a response current value in a case where a suppressing agent is not added in Example 2.

FIG. 6B is a graph showing the measurement result of a response current in the whole blood in a case where sodium azide is added as a suppressing agent in Example 2.

FIG. 6C is a graph showing the measurement result of a response current in the whole blood in a case where sodium nitrite is added as a suppressing agent in Example 2.

FIG. 7 is a graph showing the measurement result of a response current in a physiological saline in a case where sodium chloride is not added in Example 3.

FIG. 8 is a graph showing the measurement result of a response current in a physiological saline in a case where sodium chloride is added in Example 3.

DESCRIPTION OF REFERENCE SYMBOLS

-   1: concentration determination apparatus -   36: hydrogen peroxide electrode 

1. A method for determining a substrate concentration based on an amount of hydrogen peroxide generated from a substrate, wherein a suppressing agent for suppressing a reaction between the hydrogen peroxide and an inhibitor is allowed to coexist.
 2. The method for determining a substrate concentration according to claim 1, wherein the inhibitor is hemoglobin, and the suppressing agent is a compound which transforms the hemoglobin into methemoglobin.
 3. The method for determining a substrate concentration according to claim 2, wherein the suppressing agent is an azide compound.
 4. The method for determining a substrate concentration according to claim 3, wherein the azide compound is sodium azide.
 5. The method for determining a substrate concentration according to claim 3, wherein a concentration of the azide compound is 0.001 to 1.000 wt %.
 6. The method for determining a substrate concentration according to claim 2, wherein the suppressing agent is at least one selected from nitrites, ferricyanides, peroxides or permanganates.
 7. The method for determining a substrate concentration according to claim 6, wherein the suppressing agent is at least one selected from sodium nitrite, potassium ferricyanide, barium peroxide, or potassium permanganate.
 8. The method for determining a substrate concentration according to claim 1, wherein a supporting electrolyte is further allowed to coexist.
 9. The method for determining a substrate concentration according to claim 8, wherein the supporting electrolyte is sodium chloride or potassium chloride.
 10. The method for determining a substrate concentration according to claim 8, wherein a concentration of the supporting electrolyte is 0.01 to 2.00 wt %.
 11. An apparatus for determining a substrate concentration, the apparatus comprising a hydrogen peroxide electrode for determining a concentration of hydrogen peroxide generated from a substrate, wherein a suppressing agent for suppressing a reaction between the hydrogen peroxide and an inhibitor is supplied to the apparatus.
 12. The apparatus for determining a substrate concentration according to claim 11, wherein the inhibitor is hemoglobin and a compound which transforms the hemoglobin into methemoglobin is supplied as the suppressing agent.
 13. The apparatus for determining a substrate concentration according to claim 12, wherein the compound is an azide compound.
 14. The apparatus for determining a substrate concentration according to claim 13, wherein the azide compound is sodium azide.
 15. The apparatus for determining a substrate concentration according to claim 12, wherein the suppressing agent is at least one selected from nitrites, ferricyanides, peroxides or permanganates.
 16. The apparatus for determining a substrate concentration according to claim 15, wherein the suppressing agent is at least one selected from sodium nitrite, potassium ferricyanide, barium peroxide or potassium permanganate.
 17. The apparatus for determining a substrate concentration according to claim 11, wherein a supporting electrolyte is further supplied.
 18. A reagent for determining a substrate concentration based on an amount of hydrogen peroxide generated from a substrate, the reagent comprising a suppressing agent for suppressing a reaction between the hydrogen peroxide and an inhibitor.
 19. The reagent for determining a substrate concentration according to claim 18, wherein the inhibitor is hemoglobin, and the suppressing agent is a compound which transforms the hemoglobin into methemoglobin.
 20. The reagent for determining a substrate concentration according to claim 19, wherein the suppressing agent is an azide compound.
 21. The reagent for determining a substrate concentration according to claim 20, wherein the azide compound is sodium azide.
 22. The reagent for determining a substrate concentration according to claim 19, wherein the suppressing agent is at least one selected from nitrites, ferricyanides, peroxides, or permanganates.
 23. The reagent for determining a substrate concentration according to claim 22, wherein the nitrite is at least one selected from sodium nitrite, potassium ferricyanide, barium peroxide, or potassium permanganate.
 24. The reagent for determining a substrate concentration according to claim 18, further comprising a supporting electrolyte.
 25. The reagent for determining a substrate concentration according to claim 24, wherein the supporting electrolyte is sodium chloride or potassium chloride. 